JP7090288B2 - New lactic acid bacteria, innate immune activators containing new lactic acid bacteria as active ingredients, and foods and drinks containing new lactic acid bacteria - Google Patents

Description

NPMD NITE P-02308 NPMD NITE P-02307 NPMD NITE P-02306

The present invention relates to a novel lactic acid bacterium, an innate immune activator containing the novel lactic acid bacterium as an active ingredient, and a food or drink containing the novel lactic acid bacterium.

Lactic acid bacteria have long been used in fermented foods and are used in the production of foods and drinks, pharmaceuticals, probiotics, and the like. Lactic acid bacteria are characterized by being gram-positive, catalase-negative, not forming endospores, and having no motility.

In addition, lactic acid bacteria are most used as probiotics. Establishing a method for efficiently isolating highly functional lactic acid bacteria and a method for culturing these in foods are useful for the development of foods using functional lactic acid bacteria.

One of the functions that is attracting attention in probiotics is "innate immunity promoting activity". Innate immunity is at the forefront of biological defense in mammals, triggering subsequent immune responses, including antibody production. In innate immunity in mammals, various stimuli promote cytokine secretion by immunocompetent cells such as macrophages, eliminate invading pathogens, and transmit information to other immunocompetent cells.

On the other hand, invertebrates such as insects do not have acquired immunity, and pathogens are eliminated only by innate immunity. The innate immune system is known to have much in common between insects and mammals. For example, the blood cells of insects called hemosites phagocytose foreign substances that have invaded like macrophages in mammals. It is also known that Toll-like Receptors involved in the innate immune response in mammals are highly homologous to Toll Receptors involved in the innate immune response in Drosophila.

So far, the present inventors have developed an evaluation method (screening method) that can easily measure the innate immunity activation reaction in silk moths having only an innate immune mechanism (Patent Document 1, etc., Non-Patent Document 1, etc.). ). Furthermore, it has been confirmed that the method can evaluate (screening method) an innate immune activator having an innate immune activating effect on vertebrates such as humans (Patent Document 1 and the like).
Further, it has been confirmed by the present inventors that silk moth is useful as a model animal for evaluation of resistance to microbial infectious diseases (Patent Documents 2, 3, etc.).
Further, the present inventors have confirmed that the fraction purified from green tea using the muscle contraction activity of silk moth as an index has the ability to activate macrophages of mammalian cells (Non-Patent Document 2).

Abnormalities in the immune system cause a variety of diseases. Therefore, it is desired to develop an excellent innate immunity activator and a food or drink that activates innate immunity, which can regulate such an immune system desiredly.

International Publication No. 2008/126905 Japanese Unexamined Patent Publication No. 2007-327964 Japanese Unexamined Patent Publication No. 2012-006917

Ishii K, Hamamoto H, Kamimura M, Sekimizu K. Activation of the silkworm cytokine by bacterial and fungal cell wall components via a reactive oxygen species-triggered mechanism. J Biol Chem. 2008 Jan 25; 283 (4): 2185-91. Dhital, S. Purification of innate immunostimulant from green tea using a silkworm muscle contraction assay. Drug Discov. Ther. 5, 18-25 (2011).

An object of the present invention is to provide a novel lactic acid bacterium having a high innate immunity activating ability, and further, an innate immune activator containing the lactic acid bacterium or a killed bacterium or a treated product of the lactic acid bacterium as an active ingredient. It is an object of the present invention to provide a food or drink containing a lactic acid bacterium or an innate immune activator derived from the lactic acid bacterium.

As a result of diligent studies to solve the above problems, the present inventor isolated a novel lactic acid bacterium from kimchi and bran. Then, as a result of examination using a method disclosed in Patent Document 1 that can easily measure the innate immunity activation reaction, it has a higher innate immunity activation ability than the lactic acid bacteria known so far. Was confirmed.

Furthermore, as a result of analysis of the properties of the above-mentioned innate immune activating ability and analysis of the base sequence of 16S rDNA, it was found that all of them are novel lactic acid bacteria strains belonging to the genus Leuconostoc.

Further, they have found that a highly useful food can be provided by lactic acid fermentation of the lactic acid bacterium with milk, vegetable juice and fruit juice, which are not easy to purely cultivate, and have completed the present invention.

That is, the present invention provides a lactic acid bacterium belonging to the genus Leuconostoc whose accession number is NITE BP-02307 at the Patented Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE). be.

The present invention also provides a lactic acid bacterium belonging to the genus Leuconostoc whose accession number is NITE BP-02306 at the Patented Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE). be.

The present invention also provides a lactic acid bacterium belonging to the genus Leuconostoc whose accession number is NITE BP-02308 at the Patented Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE). be.

Further, the present invention is an innate immune activator containing the above-mentioned lactic acid bacterium, a dead lactic acid bacterium, or a treated product of the lactic acid bacterium as an active ingredient.
The treated product of the lactic acid bacterium is a culture of lactic acid bacterium; a concentrate; a paste; a spray-dried product, a freeze-dried product, a vacuum-dried product, a dried product such as a drum-dried product; a liquid product; a diluted product; a crushed product; a sterilized processed product. ; And; provide a natural immunostimulatory agent characterized by being at least one treated product selected from the group consisting of extracts from the culture.

The present invention also provides foods and drinks containing the above-mentioned lactic acid bacteria or the above-mentioned innate immune activator, or foods and drinks produced by using the step of fermenting with the above-mentioned lactic acid bacteria.

According to the present invention, it is possible to provide a novel lactic acid bacterium having a higher innate immune activation ability than the lactic acid bacteria known so far.
Further, a natural immune activator containing the lactic acid bacterium as an active ingredient, a food or drink containing the lactic acid bacterium or the innate immune activator, and a food or drink produced by a step of fermenting the lactic acid bacterium. Goods can be provided.

Further, the lactic acid bacterium of the present invention can be used not only for the production of general foods and drinks, health foods, drugs, fermented foods and drinks, probiotics, etc., but also for the prevention and treatment of diseases by activating natural immunity. .. In addition, because it is resistant to acid, it is not decomposed in the stomach and easily reaches the small intestine.

Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified within the scope of the technical idea.

<Lactic acid bacteria # 7-2>
The lactic acid bacterium of the present invention is a lactic acid bacterium belonging to the genus Leuconostoc whose accession number is NITE BP-02307 at the Patented Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE). It may be abbreviated as "lactic acid bacteria # 7-2").

Hereinafter, a novel lactic acid bacterium strain (lactic acid bacterium # 7-2) belonging to the genus Leuconostoc will be described in detail.
The lactic acid bacterium # 7-2 of the present invention was isolated for the first time using kimchi as an isolation source.

Gram stain result: Positive Bacterial shape: Spherical Aerobic / anaerobic: Anaerobic Lactic acid-producing ability: Yes

Physiological properties: The physiological and chemical taxonomic properties of the lactic acid bacterium # 7-2 of the present invention are as follows.
(1) Catalase:-
(2) Acid phosphatase: +
(3) Alkaline phosphatase: +
(4) Naphthol-AS-BI-phosphohydrolase: +
(5) Esterase (C4): +
(6) α-galactosidase:-
(7) Esterase lipase (C8): +
(8) β-galactosidase:-
(9) Lipase (C14):-
(10) β-glucuronidase:-
(11) Leucine allyl amidase: +
(12) α-Glucosidase: +
(13) Valine allyl amidase:-
(14) β-Glucosidase:-
(15) Cystine allyl amidase:-
(16) N-Acetyl-β-Glucosaminidase:-
(17) Trypsin:-
(18) α-Mannosidase:-
(19) α-chymotrypsin:-
(20) α-fucosidase:-

(21) Ability to generate acid and gas from the following sugars, etc. Glycerol:-
Erythritol:-
D-Arabinose:-
L-Arabinose:-
D-Ribose: +
D-Xylose:-
L-Xylose:-
D-Adonitol:-
β-Methyl-D-xylopyranoside:-
D-Galactose: +
D-Glucose: +
D-Fructose: +
D-Mannose: +
L-Sorbose:-
L-Rhamnose:-
Dulcitol:-
Inositol:-
D-Mannitol:-
D-Sorbitol:-
α-Methyl-D-mannopyranoside:-
α-Methyl-D-glucopyranoside: +
N-Acetyl glucosamine: +
Amygdalin: +
Arbutin: +
Esculin ferric citrate: +
Salicin: +
D-Cellobiose: +
D-Maltose: +
D-Lactose:-
D-Melibiose: +
D-Sucrose: +
D-Trehalose: +
Insulin:-
D-Melezitose: +
D-Raffinose: +
Starch: +
Glycogen:-
Xylitol:-
Gentiobiose: +
D-Turanose: +
D-Lyxose:-
D-Tagatose: +
D-Fucose:-
L-Fucose:-
D-Arabitol:-
L-Arabitol:-
Gluconate: +
2-Keto-gluconate:-
5-Keto-gluconate:-

Molecular Biological Analysis Results: The analysis results of Lactobacillus # 7-2 regarding 16SrDNA used as an index of molecular biological phylogenetic classification are as shown in SEQ ID NO: 1 in the attached sequence listing.
That is, as a result of amplifying the base sequence of the 16SrDNA region by PCR from the genomic DNA of lactic acid bacterium # 7-2 and analyzing it with a sequencer, a base sequence corresponding to almost the entire length of 16SrDNA was found.
When this base sequence was subjected to a homology search by BLAST analysis of NCBI, the base sequence of the 16SrDNA region of lactic acid bacterium # 7-2 was the base sequence of the Leuconostoc carnosum JB16 strain belonging to the genus Leuconostoc (registration number: NR_102781.1). ) And 99% homology, lactic acid bacterium # 7-2 belongs to Leuconostoc carnosum.
However, the lactic acid bacterium # 7-2 of the present invention is a lactic acid bacterium strain different from the above-mentioned strain because it does not completely match even when only the 16SrDNA regions are compared.

The physiological and chemical taxonomic properties of the above-mentioned lactic acid bacterium # 7-2 are compared with the classification by Bergey's Manual of Systematic Bacteriology (vol.3 1989) and the contents of other literatures. In addition, as a result of judgment in consideration of the result of the above 16SrDNA analysis, "lactic acid bacterium # 7-2" of the present invention is a novel microorganism belonging to the genus Leuconostoc.
In addition, there is no microorganism having a base sequence of 16SrDNA region that matches the base sequence of 16SrDNA region of lactic acid bacterium # 7-2, and it exhibits a higher innate immune activation effect as compared with known strains belonging to the genus Leuconostocaceae. As a result of comprehensive examination including the above, it was judged that lactic acid bacterium # 7-2 was an isolated novel microbial strain.

Lactic acid bacteria # 7-2 is a patented microorganism of the National Institute of Technology and Evaluation (hereinafter abbreviated as "NITE"), Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture. It is a microorganism that has been deposited domestically at the Deposit Center (NPMD) and has been deposited under the deposit number: NITE P-02307 (deposit date: July 26, 2016).
After that, Lactobacillus # 7-2 submitted an original deposit application to the Patent Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE), Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture. After submitting, apply for transfer from domestic deposit (original deposit date: July 26, 2016) to deposit based on the Butapest Treaty (transfer date (international deposit date): July 25, 2017), and survive. As a result of receiving the application for transfer to the deposit (international deposit) based on the Butapest Treaty, the deposit number "NITE BP-02307" has been received.

As a general property of a bacterium, the property as a strain is easily mutated, so that the lactic acid bacterium # 7-2 of the present invention may not stay within the range of the physiological properties shown above. It goes without saying that such "mutations" include both natural and artificial mutations.

The method for culturing lactic acid bacteria # 7-2 will be described below. The culturing method of lactic acid bacterium # 7-2 may be carried out according to a general culturing method performed on a microorganism of the genus Leuconostocaceae.
Culturing is preferably carried out under anaerobic conditions. Examples of carbon sources in the medium include D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, amigdalin, arbutin, esculin, salicin, and D-cellobiose. , D-maltose, schucrose, D-trehalose, gentiobiose, sugar syrup, water candy, oils and fats and other organic carbon compounds are used, and the nitrogen source is meat extract, casein, peptone, yeast extract, dried yeast, germ, soybean. Organic / inorganic nitrogen compounds such as powder, urea, amino acids, and ammonium salts can be used.
As the salts, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt, phosphate, iron salt, copper salt, zinc salt and cobalt salt are appropriately added as needed. Further, it is preferable to add a growth promoting substance such as biotin, vitamin B1, cystine, methyl oleate, lard oil and the like in terms of increasing the production amount of the target product.
Further, a defoaming agent such as silicone oil or a surfactant may be added. As the prepared medium, for example, MRS medium, GAM medium and the like are preferably used.

As described above, the culture conditions may be the same as the general culture conditions performed for the microorganisms of the genus Leuconostocaceae. If it is a liquid culture method, static culture is desirable. If the scale is small, a static culture method using a glass bottle with a lid may be used.
The culture temperature is preferably maintained between 25 ° C. and 37 ° C., and more preferably 30 ° C. to 37 ° C. The culture pH is preferably around 7. The culture period is a factor that varies depending on the medium composition used, the culture temperature, etc., but in the case of lactic acid bacterium # 7-2, a sufficient amount of the target substance is preferably 12 to 72 hours, more preferably 24 to 48 hours. Can be secured.
It is also preferable to pick up the colonies obtained by culturing and perform single colonization on the medium again.

<Lactic acid bacteria # 4-2>
The lactic acid bacterium of the present invention is a lactic acid bacterium belonging to the genus Leuconostoc, which is NITE BP-02306 at the Patented Microbial Deposit Center (NPMD) of the National Institute of Technology and Evaluation (NITE) (hereinafter, "lactic acid bacterium #". 4-2 "may be abbreviated).

Hereinafter, a novel lactic acid bacterium strain (lactic acid bacterium # 4-2) belonging to the genus Leuconostoc will be described in detail.
Lactic acid bacterium # 4-2 of the present invention was isolated for the first time using bran as an isolation source.

Gram stain result: Positive Bacterial shape: Spherical Aerobic / anaerobic: Anaerobic Lactic acid-producing ability: Yes

Physiological properties: The physiological and chemical taxonomic properties of the lactic acid bacterium # 4-2 of the present invention are as follows.
(1) Catalase:-
(2) Acid phosphatase: +
(3) Alkaline phosphatase:-
(4) Naphthol-AS-BI-phosphohydrolase: +
(5) Esterase (C4): +
(6) α-galactosidase:-
(7) Esterase lipase (C8): +
(8) β-galactosidase:-
(9) Lipase (C14):-
(10) β-glucuronidase:-
(11) Leucine allyl amidase: +
(12) α-Glucosidase: +
(13) Valine allyl amidase:-
(14) β-Glucosidase: +
(15) Cystine allyl amidase:-
(16) N-Acetyl-β-Glucosaminidase:-
(17) Trypsin:-
(18) α-Mannosidase:-
(19) α-chymotrypsin: +
(20) α-fucosidase:-

(21) Ability to generate acid and gas from the following sugars, etc. Glycerol:-
Erythritol:-
D-Arabinose:-
L-Arabinose: +
D-Ribose: +
D-Xylose: +
L-Xylose:-
D-Adonitol:-
β-Methyl-D-xylopyranoside:-
D-Galactose: +
D-Glucose: +
D-Fructose: +
D-Mannose: +
L-Sorbose:-
L-Rhamnose:-
Dulcitol:-
Inositol:-
D-Mannitol: +
D-Sorbitol:-
α-Methyl-D-mannopyranoside:-
α-Methyl-D-glucopyranoside: +
N-Acetyl glucosamine: +
Amygdalin: +
Arbutin: +
Esculin ferric citrate: +
Salicin: +
D-Cellobiose: +
D-Maltose: +
D-Lactose:-
D-Melibiose: +
D-Sucrose: +
D-Trehalose: +
Insulin:-
D-Melezitose: +
D-Raffinose:-
Starch: +
Glycogen:-
Xylitol:-
Gentiobiose: +
D-Turanose: +
D-Lyxose:-
D-Tagatose: +
D-Fucose:-
L-Fucose:-
D-Arabitol:-
L-Arabitol:-
Gluconate: +
2-Keto-gluconate: +
5-Keto-gluconate:-

Molecular Biological Analysis Results: The analysis results of Lactobacillus # 4-2 regarding 16SrDNA used as an index of molecular biological phylogenetic classification are as shown in SEQ ID NO: 2 in the attached sequence listing.
That is, as a result of amplifying the base sequence of the 16SrDNA region by PCR from the genomic DNA of lactic acid bacterium # 4-2 and analyzing it with a sequencer, a base sequence corresponding to almost the entire length of 16SrDNA was found.
When this base sequence was subjected to a homology search by BLAST analysis of NCBI, the base sequence of the 16SrDNA region of lactic acid bacterium # 4-2 was the base sequence of the Leuconostoc gelidum POUF4d strain belonging to the genus Leuconostoc (registration number: NR_133769.1). ) And 99% homology, lactic acid bacterium # 4-2 belongs to Leuconostoc gelidum.
However, the lactic acid bacterium # 4-2 of the present invention is a lactic acid bacterium strain different from the above-mentioned strain because it does not completely match even when only the 16SrDNA regions are compared.

The physiological and chemical taxonomic properties of the above-mentioned lactic acid bacterium # 4-2 are compared with the classification by Bergey's Manual of Systematic Bacteriology (vol.3 1989) and the contents of other literatures. In addition, as a result of judgment in consideration of the result of the above 16S rDNA analysis, "lactic acid bacterium # 4-2" of the present invention is a novel microorganism belonging to the genus Leuconostoc.
In addition, there is no microorganism having a base sequence of 16SrDNA region that matches the base sequence of 16SrDNA region of lactic acid bacterium # 4-2, and it exhibits a higher innate immune activation effect as compared with known strains belonging to the genus Leuconostocaceae. As a result of comprehensive examination including the above, it was judged that lactic acid bacterium # 4-2 was an isolated novel microbial strain.

Lactic acid bacteria # 4-2 is a patented microorganism of the National Institute of Technology and Evaluation (hereinafter abbreviated as "NITE"), Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture. It is a microorganism that has been deposited domestically at the Deposit Center (NPMD) and has been deposited under the deposit number: NITE P-02306 (deposit date: July 26, 2016).
After that, Lactobacillus # 4-2 submitted an original deposit application to the Patent Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE), Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture. After submitting, apply for transfer from domestic deposit (original deposit date: July 26, 2016) to deposit based on the Butapest Treaty (transfer date (international deposit date): July 25, 2017), and survive. As a result of receiving the application for transfer to the deposit (international deposit) based on the Butapest Treaty, the deposit number "NITE BP-02306" has been received.

As a general property of a bacterium, the property as a strain is easily mutated, so that the lactic acid bacterium # 4-2 of the present invention may not stay within the range of the physiological properties shown above. It goes without saying that such "mutations" include both natural and artificial mutations.

The method for culturing lactic acid bacteria # 4-2 will be described below. The culturing method of lactic acid bacterium # 4-2 may be carried out according to a general culturing method performed on a microorganism of the genus Leuconostocaceae.
Culturing is preferably carried out under anaerobic conditions. Examples of carbon sources in the medium include D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, amigdalin, arbutin, esculin, salicin, and D-cellobiose. , D-maltose, schucrose, D-trehalose, gentiobiose, sugar syrup, water candy, oils and fats and other organic carbon compounds are used, and the nitrogen source is meat extract, casein, peptone, yeast extract, dried yeast, germ, soybean. Organic / inorganic nitrogen compounds such as powder, urea, amino acids, and ammonium salts can be used.
As the salts, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt, phosphate, iron salt, copper salt, zinc salt and cobalt salt are appropriately added as needed. Further, it is preferable to add a growth promoting substance such as biotin, vitamin B1, cystine, methyl oleate, lard oil and the like in terms of increasing the production amount of the target product.
Further, a defoaming agent such as silicone oil or a surfactant may be added. As the prepared medium, for example, MRS medium, GAM medium and the like are preferably used.

As described above, the culture conditions may be the same as the general culture conditions performed for the microorganisms of the genus Leuconostocaceae. If it is a liquid culture method, static culture is desirable. If the scale is small, a static culture method using a glass bottle with a lid may be used.
The culture temperature is preferably maintained between 25 ° C. and 37 ° C., and more preferably 30 ° C. to 37 ° C. The culture pH is preferably around 7. The culture period is a factor that varies depending on the medium composition used, the culture temperature, etc., but in the case of lactic acid bacterium # 4-2, a sufficient amount of the target substance is preferably 12 to 72 hours, more preferably 24 to 48 hours. Can be secured.
It is also preferable to pick up the colonies obtained by culturing and perform single colonization on the medium again.

<Lactic acid bacteria 8 / 11-3>
The lactic acid bacterium of the present invention is a lactic acid bacterium belonging to the genus Leuconostoc whose accession number is NITE BP-02308 at the Patented Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE). It may be abbreviated as "lactic acid bacteria 8 / 11-3").

Hereinafter, a novel lactic acid bacterium strain (lactic acid bacterium 8 / 11-3) belonging to the genus Leuconostoc will be described in detail.
The lactic acid bacterium 8 / 11-3 of the present invention was isolated for the first time using kimchi as an isolation source.

Gram stain result: Positive Bacterial shape: Spherical Aerobic / anaerobic: Anaerobic Lactic acid-producing ability: Yes

Physiological properties: The physiological and chemical taxonomic properties of the lactic acid bacterium 8 / 11-3 of the present invention are as follows.
(1) Catalase:-
(2) Acid phosphatase: +
(3) Alkaline phosphatase: +
(4) Naphthol-AS-BI-phosphohydrolase: +
(5) Esterase (C4): +
(6) α-galactosidase: +
(7) Esterase lipase (C8): +
(8) β-galactosidase: +
(9) Lipase (C14):-
(10) β-glucuronidase:-
(11) Leucine allyl amidase: +
(12) α-Glucosidase: +
(13) Valine allyl amidase:-
(14) β-Glucosidase: +
(15) Cystine allyl amidase:-
(16) N-Acetyl-β-Glucosaminidase:-
(17) Trypsin:-
(18) α-Mannosidase:-
(19) α-chymotrypsin:-
(20) α-fucosidase:-

(21) Ability to generate acid and gas from the following sugars, etc. Glycerol:-
Erythritol:-
D-Arabinose:-
L-Arabinose: +
D-Ribose: +
D-Xylose: +
L-Xylose:-
D-Adonitol:-
β-Methyl-D-xylopyranoside:-
D-Galactose: +
D-Glucose: +
D-Fructose: +
D-Mannose: +
L-Sorbose:-
L-Rhamnose:-
Dulcitol:-
Inositol:-
D-Mannitol: +
D-Sorbitol: +
α-Methyl-D-mannopyranoside:-
α-Methyl-D-glucopyranoside: +
N-Acetyl glucosamine: +
Amygdalin: +
Arbutin: +
Esculin ferric citrate: +
Salicin: +
D-Cellobiose: +
D-Maltose: +
D-Lactose: +
D-Melibiose: +
D-Sucrose: +
D-Trehalose: +
Insulin:-
D-Melezitose: +
D-Raffinose: +
Starch:-
Glycogen:-
Xylitol:-
Gentiobiose: +
D-Turanose: +
D-Lyxose:-
D-Tagatose: +
D-Fucose:-
L-Fucose:-
D-Arabitol:-
L-Arabitol:-
Gluconate: +
2-Keto-gluconate:-
5-Keto-gluconate:-

Molecular Biological Analysis Results: The analysis results of Lactobacillus 8 / 11-3 regarding 16SrDNA used as an index of molecular biological phylogenetic classification are as shown in SEQ ID NO: 3 in the attached sequence listing.
That is, as a result of amplifying the base sequence of the 16SrDNA region by PCR from the genomic DNA of lactic acid bacterium 8 / 11-3 and analyzing it with a sequencer, a base sequence corresponding to almost the entire length of 16SrDNA was found.
When this base sequence was subjected to a homology search by BLAST analysis of NCBI, the base sequence of the 16SrDNA region of lactic acid bacteria 8 / 11-3 was the base sequence of the Leuconostoc mesenteroides ATCC8293 strain belonging to the genus Leuconostoc (registration number: NR_074957. Since it showed 99% homology with 1), lactic acid bacterium 8 / 11-3 belongs to Leuconostoc mesenteroides.
However, the lactic acid bacterium 8 / 11-3 of the present invention is a lactic acid bacterium strain different from the above-mentioned strain because it does not completely match even when comparing only the 16SrDNA regions.

The physiological and chemical taxonomic properties of the above-mentioned lactic acid bacterium 8 / 11-3 are classified by Bergey's Manual of Systematic Bacteriology (vol.3 1989) and described in other documents. As a result of comparison and further, as a result of judgment in consideration of the result of the above 16SrDNA analysis, "lactic acid bacterium 8 / 11-3" of the present invention is a novel microorganism belonging to the genus Leuconostoc.
In addition, there is no microorganism having a base sequence of 16SrDNA region that matches the base sequence of 16SrDNA region of lactic acid bacteria 8 / 11-3, and it shows a high innate immune activity as compared with known strains belonging to the genus Leuconostoc. As a result of comprehensive examination including the above, it was judged that lactic acid bacterium 8 / 11-3 was an isolated novel microbial strain.

Lactic acid bacteria 8 / 11-3 is a patent of National Institute of Technology and Evaluation (hereinafter abbreviated as "NITE"), Room 2-5-8 122 Kazusakamatari, Kisarazu City, Chiba Prefecture. It is a microorganism that has been deposited domestically at the Microbial Deposit Center (NPMD) and has been deposited under the deposit number: NITE P-02308 (deposit date: July 26, 2016).
Lactobacillus 8 / 11-3 was subsequently submitted to the Patent Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE) in Room 2-5-8 122 Kazusakamatari, Kisarazu City, Chiba Prefecture. And applied for transfer from domestic deposit (original deposit date: July 26, 2016) to deposit based on the Butapest Treaty (transfer date (international deposit date): May 22, 2017) and survived. As a result of receiving the transfer application to the deposit (international deposit) based on the Butapest Treaty, the deposit number "NITE BP-02308" has been received.

As a general property of a bacterium, the property as a strain is easily mutated, so that the lactic acid bacterium 8 / 11-3 of the present invention may not stay within the range of the physiological properties shown above. .. It goes without saying that such "mutations" include both natural and artificial mutations.

The method for culturing lactic acid bacteria 8 / 11-3 will be described below. The method for culturing lactic acid bacteria 8 / 11-3 may be carried out according to a general culture method for microorganisms of the genus Leuconostocaceae.
Culturing is preferably carried out under anaerobic conditions. Examples of carbon sources in the medium include D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, amigdalin, arbutin, esculin, salicin, and D-cellobiose. , D-maltose, schucrose, D-trehalose, gentiobiose, sugar syrup, water candy, oils and fats and other organic carbon compounds are used, and the nitrogen source is meat extract, casein, peptone, yeast extract, dried yeast, germ, soybean. Organic / inorganic nitrogen compounds such as powder, urea, amino acids, and ammonium salts can be used.
As the salts, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt, phosphate, iron salt, copper salt, zinc salt and cobalt salt are appropriately added as needed. Further, it is preferable to add a growth promoting substance such as biotin, vitamin B1, cystine, methyl oleate, lard oil and the like in terms of increasing the production amount of the target product.
Further, a defoaming agent such as silicone oil or a surfactant may be added. As the prepared medium, for example, MRS medium, GAM medium and the like are preferably used.

As described above, the culture conditions may be the same as the general culture conditions performed for the microorganisms of the genus Leuconostocaceae. If it is a liquid culture method, static culture is desirable. If the scale is small, a static culture method using a glass bottle with a lid may be used.
The culture temperature is preferably maintained between 25 ° C. and 37 ° C., and more preferably 30 ° C. to 37 ° C. The culture pH is preferably around 7. The culture period is a factor that varies depending on the medium composition used, the culture temperature, etc., but in the case of lactic acid bacteria 8 / 11-3, a sufficient amount of the target product is preferably 12 to 72 hours, more preferably 24 to 48 hours. Can be secured.
It is also preferable to pick up the colonies obtained by culturing and perform single colonization on the medium again.

<Innate immune activator>
The innate immune activator of the present invention is an innate immune activator containing the above-mentioned lactic acid bacterium, a killed lactic acid bacterium, or a treated product of the lactic acid bacterium as an active ingredient.
The treated product of the above lactic acid bacterium is at least selected from the group consisting of a culture, a concentrate, a paste, a dried product, a liquefied product, a diluted product, a crushed product, a sterilized processed product, and an extract from the culture. It is characterized by being one processed product.

The innate immune activator of the present invention can contain the above-mentioned lactic acid bacterium of the present invention, a dead lactic acid bacterium, or a treated product of the lactic acid bacterium in various states. For example, a state containing a suspension, a lactic acid bacterium cell, a culture supernatant, a medium component, and the like can be mentioned.

The innate immune activator of the present invention may contain the above-mentioned lactic acid bacterium as it is, or may be contained as a lactic acid bacterium-treated product obtained by subjecting the lactic acid bacterium to some treatment.
Examples of the treated product of lactic acid bacteria used for the natural immunostimulatory agent include cultures of lactic acid bacteria; concentrates; pastes; spray-dried products, freeze-dried products, vacuum-dried products, dried products such as drum-dried products; liquids. Examples thereof include a product; a diluted product; a crushed product; a sterilized processed product; an extract from the culture; and the like.

As the lactic acid bacteria, live cells, wet bacteria, dried bacteria and the like can be appropriately used. Further, it may be a killed bacterium that has been sterilized, that is, has undergone heat sterilization treatment, radiation sterilization treatment, crushing treatment, or the like.

The content of lactic acid bacteria, killed bacteria of the lactic acid bacteria, and treated products of the lactic acid bacteria, which are the active ingredients in the natural immunostimulatory agent of the present invention, with respect to the entire natural immune activator is not particularly limited and may be used according to the purpose. It can be appropriately selected, but when the total amount of the natural immune activator is 100 parts by mass, the total amount of lactic acid bacteria, killed bacteria of the lactic acid bacteria, and treated product of the lactic acid bacteria is 0.001 to 100 parts by mass. It is preferably contained in, more preferably 0.01 to 99 parts by mass, particularly preferably 0.1 to 95 parts by mass, and further preferably 1 to 90 parts by mass.

In addition, any one of the above active ingredients may be used alone, or two or more thereof may be used in combination. When two or more kinds are used in combination, the content ratio of each active ingredient in the innate immune activator is not particularly limited and can be appropriately selected according to the purpose.

The innate immune activator of the present invention contains the above-mentioned lactic acid bacteria, the above-mentioned killed bacteria of the above-mentioned lactic acid bacteria, or the treated product of the above-mentioned lactic acid bacteria as an active ingredient, but can contain "other ingredients" in addition to these active ingredients. ..
The "other components" in the innate immune activator are not particularly limited and can be appropriately selected depending on the intended purpose within the range not impairing the effect of the present invention, and are pharmaceutically acceptable, for example. Examples thereof include carriers that can be used.
The carrier is not particularly limited and may be appropriately selected depending on, for example, a dosage form described later. Further, the content of "other components" in the innate immune activator is not particularly limited and may be appropriately selected depending on the intended purpose.

<Pharmaceuticals; Food and drink; Health foods, etc.>
The lactic acid bacterium of the present invention and the natural immunostimulatory agent of the present invention derived from the lactic acid bacterium include foods and drinks having specifications such as pharmaceuticals (pharmaceuticals), quasi-drugs, general foods and drinks, health foods, fermented foods and drinks, and powdered milk. It can be blended into various medicines, foods and drinks, etc. regardless of their form. It can also be used for the production of probiotics.
Above all, foods and drinks produced by using the above-mentioned step of fermenting with the lactic acid bacterium of the present invention, and among them, fermented milk is easy to exert the usual effect of the lactic acid bacterium and the above-mentioned effect peculiar to the present invention. preferable.

The dosage form of the innate immune activator of the present invention is not particularly limited, and can be appropriately selected, for example, according to a desired administration method as described later.
Specifically, for example, oral solid preparations (tablets, coated tablets, granules, powders, hard capsules, soft capsules, etc.), oral liquid preparations (oral liquid preparations, syrup preparations, elixir preparations, etc.), injections (solvents, suspensions, etc.) Agents, etc.), ointments, patches, gels, creams, external powders, sprays, inhalation sprays, etc.

As the oral solid preparation, for example, an excipient and, if necessary, an additive such as a binder, a disintegrant, a lubricant, a colorant, a flavoring / odorant, etc. are added to the active ingredient, and a conventional method is used. Can be manufactured by

Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like.
Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac, calcium phosphate, polyvinylpyrrolidone and the like. Be done.
Examples of the disintegrant include dried starch, sodium alginate, canten powder, sodium hydrogencarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose and the like.
Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol and the like.
Examples of the colorant include titanium oxide, iron oxide and the like.
Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.

The oral solution can be produced by a conventional method, for example, by adding an additive such as a taste-masking agent, a buffering agent, a stabilizer, an edible (processed) oil, or an animal or vegetable oil to the active ingredient.

Examples of the taste-masking agent include sucrose, orange peel, citric acid, tartaric acid and the like. Examples of the buffering agent include sodium citrate and the like. Examples of the stabilizer include tragant, gum arabic, gelatin and the like.

As the injection, for example, a pH adjuster, a buffer, a stabilizer, an tonicity agent, a local anesthetic, etc. are added to the active ingredient, and the injection is subcutaneously, intramuscularly, or intravenously used by a conventional method. Etc. can be produced.
Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium metabisulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of the tonicity agent include sodium chloride, glucose and the like. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride and the like.

As the ointment, for example, a known base, stabilizer, wetting agent, preservative and the like can be blended with the active ingredient and mixed by a conventional method to produce the ointment.
Examples of the base include liquid paraffin, white petrolatum, bleached beeswax, octyldodecyl alcohol, paraffin and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like.

As the patch, for example, a cream, a gel, a paste or the like as an ointment can be applied to a known support by a conventional method to produce the patch. Examples of the support include cotton, rayon, woven fabric made of chemical fibers, non-woven fabric, soft vinyl chloride, polyethylene, polypropylene, polyurethane and other films, foam sheets and the like.

The innate immune activator of the present invention can be suitably used, for example, for individuals, bacteria and the like that require activation of the innate immune system.
Specifically, for example, by administering to an individual who needs to maintain health or recover from fatigue; an individual who needs prevention or treatment of cancer or lifestyle-related diseases; an individual infected with bacteria, fungi, viruses, etc. Can be used.

The target animal for administration of the natural immunostimulatory agent of the present invention is not particularly limited, but for example, humans; experimental animals such as mice and rats; monkeys; horses; domestic animals such as cows, pigs, goats and chickens; cats, Pets such as dogs; etc.

The method for administering the innate immune activator is not particularly limited and may be appropriately selected depending on, for example, the above-mentioned dosage form, such as oral administration, intraperitoneal administration, injection into blood, and intestine. Injection into the inside and the like can be mentioned. Of these, oral administration is preferable because it is simple and exerts the above effects, and oral administration as foods and drinks such as general foods and drinks, health foods, and fermented foods and drinks is particularly preferable.

The dose of the innate immune activator is not particularly limited or limited, and can be appropriately selected depending on the age, body weight, degree of desired effect, etc. of the individual to be administered. For example, to an adult. The daily dose of the active ingredient is preferably 1 mg to 30 g, more preferably 10 mg to 10 g, and particularly preferably 100 mg to 3 g.
Further, the administration time is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it may be administered prophylactically or therapeutically.

"Foods and drinks containing the above-mentioned lactic acid bacteria of the present invention, killed or treated products of the lactic acid bacteria, natural immune activators, and foods and drinks produced by a step of fermenting with the lactic acid bacteria" (hereinafter, in parentheses). The content of lactic acid bacteria and the natural immune activator in (may be simply abbreviated as "food and drink of the present invention") is not particularly limited, and is appropriate according to the purpose and the mode (type) of the food and drink. Although it can be selected, when the total amount of food and drink is 100 parts by mass, the total amount is preferably 0.001 to 100 parts by mass, more preferably 0.01 to 99 parts by mass. Particularly preferably, the content is 0.1 to 95 parts by mass.

Further, any one of the above may be used alone, or two or more thereof may be used in combination. When two or more kinds are used in combination, the content ratio of each substance in the above-mentioned food and drink is not particularly limited and can be appropriately selected according to the purpose.

The food and drink of the present invention has an innate immune activation ability and / or an infectious disease preventive treatment ability.
The food or drink of the present invention may further contain "other components" in addition to the above-mentioned innate immune activator and infectious disease preventive and therapeutic agent of the present invention.

The above-mentioned "other ingredients" are not particularly limited and may be appropriately selected depending on the intended purpose within the range not impairing the effect of the present invention, and examples thereof include various food raw materials. The content of the "other components" is not particularly limited and may be appropriately selected depending on the intended purpose.

The lactic acid bacterium of the present invention can be used for the production of general foods and drinks, health foods, drugs, fermented foods and drinks, probiotics and the like. As fermented foods and drinks, it is particularly suitable for use as fermented milk, lactic acid bacteria beverages, yogurt, pickles, lactic acid bacteria starters for producing pickles, and the like.

The type of the above food is not particularly limited and may be appropriately selected depending on the purpose. For example, confectioneries such as jelly, candy, chocolate, biscuits, and gummy; tastes such as green tea, tea, coffee, and soft drinks. Beverages; dairy products such as fermented milk, yogurt, ice cream, lacto ice; vegetable beverages, fruit beverages, processed vegetables and fruits such as jams; liquid foods such as soups; processed grain products such as breads and noodles; various seasonings Fees; etc. Among them, dairy products such as yogurt and fermented milk are preferable.
The method for producing these foods is not particularly limited, and for example, they can be appropriately produced according to ordinary methods for producing various foods.

Further, the above-mentioned food may be produced as, for example, an oral solid preparation such as a tablet, a granule or a capsule, or an oral liquid preparation such as an internal liquid preparation or a syrup preparation. The method for producing the oral solid preparation and the oral liquid preparation is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the oral solid preparation and the oral liquid preparation can be manufactured according to the above-mentioned method for producing the oral solid preparation and the oral liquid preparation. can.

The food and drink of the present invention is particularly useful as a functional food, a health food, etc. for the purpose of activating the innate immune system and imparting resistance to infectious diseases.
When the lactic acid bacterium of the present invention, the dead bacterium, the treated product, or the like is used for producing a food or drink, the production method can be carried out by a method well known to those skilled in the art. If you are a person skilled in the art, a step of mixing the (dead) cell or processed product of the lactic acid bacterium of the present invention with other components, a molding step, a sterilization step, a fermentation step, a baking step, a drying step, a cooling step, a granulation step, It is possible to make the desired food and drink by appropriately combining the packaging processes and the like.

Further, when the lactic acid bacterium of the present invention is used for producing various fermented milks, it can be produced by a method well known to those skilled in the art. For example, foods and drinks produced by using the step of adding a required amount of the lactic acid bacterium of the present invention to fermented milk as a dead bacterium, and food and drink manufactured by using the step of fermenting the lactic acid bacterium of the present invention as a lactic acid bacterium starter. Can be mentioned.
When fermentation is carried out using the lactic acid bacterium of the present invention as the lactic acid bacterium starter, it can be carried out under the same conditions as the culture conditions of the lactic acid bacterium of the present invention.

Hereinafter, the present invention will be described in more detail based on Examples and Study Examples, but the present invention is not limited to the specific scope of the following Examples and the like.

As described above, "lactic acid bacteria # 7-2" and "lactic acid bacteria 8 / 11-3" used in the examples were isolated from kimchi, and "lactic acid bacteria # 4-2" were isolated from bran.

Lactic acid bacteria # 7-2 belonging to Leuconostoc carnosum are located at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (Kazusakamatari Room 2-5-8, Kisarazu City, Chiba Prefecture). It has been deposited (deposit number: NITE P-02307, deposit date July 26, 2016).
After that, Lactobacillus # 7-2 submitted an original deposit application to the Patent Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE), Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture. After submitting, apply for transfer from domestic deposit (original deposit date: July 26, 2016) to deposit based on the Butapest Treaty (transfer date (international deposit date): July 25, 2017), and survive. As a result of receiving the application for transfer to the deposit (international deposit) based on the Butapest Treaty, the deposit number "NITE BP-02307" has been received.

Lactic acid bacteria # 4-2 belonging to Leuconostoc gelidum are located at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (Kazusakamatari Room 2-5-8, Kisarazu City, Chiba Prefecture). It has been deposited (deposit number: NITE P-02306, deposit date July 26, 2016).
Similar to the above-mentioned lactic acid bacterium # 7-2, lactic acid bacterium # 4-2 also submits the original deposit application to the Patented Microorganisms Deposit Center (NPMD), and from the domestic deposit (original deposit date: July 26, 2016). An application for transfer to a deposit under the Butapest Treaty was submitted (transfer date (international deposit date): July 25, 2017), survival was proved, and an application for transfer to a deposit under the Butapest Treaty (international deposit) was received. As a result, the contract number "NITE BP-02306" has been received.

Lactic acid bacteria 8 / 11-3 belonging to Leuconostoc mesenteroides are the Patented Microbial Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NPMD) (Room 122, Kazusakamatari, Kisarazu City, Chiba Prefecture). (Deposit number: NITE P-02308, deposit date July 26, 2016).
Similar to Lactic Acid Bacteria # 7-2, Lactic Acid Bacteria 8 / 11-3 also submitted the original deposit application to the Patent Microorganisms Deposit Center (NPMD), and from the domestic deposit (original deposit date: July 26, 2016). , Submitted an application for transfer to a deposit under the Butapest Treaty (transfer date (international deposit date): May 22, 2017), proved survival, and received an application for transfer to a deposit under the Butapest Treaty (international deposit) As a result, the contract number "NITE BP-02308" has been received.

<Materials and methods>
<< Measurement of innate immunity promoting activity of lactic acid bacteria using silk moth muscle specimen >>
The innate immunity promoting activity was measured by using the method described in Patent Document 1 and using the silk moth muscle contraction activity as an index. Lactic acid bacteria were spread on 0.5% calcium carbonate MRS agar medium and anaerobically cultured at 30 ° C. to form colonies. Colonies were collected, inoculated into 14 mL of MRS medium added to a 15 mL tube, and statically cultured at 30 ° C. for 2 days. The entire amount of this culture solution was added to 100 mL of MRS liquid medium, and the cells were statically cultured at 30 ° C. for 1 day. The culture was autoclaved at 121 ° C for 20 minutes and centrifuged at 8000 rpm at 4 ° C for 10 minutes. The precipitate was washed with 50 mL of 0.9% NaCl and then centrifuged again at 8000 rpm at 4 ° C. for 10 minutes, and the precipitate was suspended in 1 mL of 0.9% NaCl solution. 50 μL of this suspension was diluted and injected into a silk moth muscle specimen, and the change in length of the silk moth specimen before and after injection was measured. The sample amount in which the length of the silk moth muscle specimen was reduced by 15% was defined as 1 unit. On the other hand, 100 μL of the suspension of the bacterium was dried by a centrifugal evaporator, and the dry weight was measured.

<< Proliferation test of each lactic acid bacterium in milk, fruit and vegetable juice >>
Preculture of lactic acid bacteria was performed by anaerobic culture at 30 ° C. using MRS liquid medium. A preculture solution was added to 50 mL of various vegetable / fruit juices, and static culture was carried out at 30 ° C. The viable cell count of lactic acid bacteria was measured by spreading the culture solution on an MRS agar medium, anaerobically culturing at 30 ° C., and measuring the number of colonies that appeared. When it became 1.0 × 10 ⁶ cells / mL or more, it was judged that the proliferative ability was “presence”.

The bitter gourd juice was prepared by putting bitter gourd in RO water, treating it with a juicer, and autoclaving it at 121 ° C. for 20 minutes.
Kiwi juice was prepared by adding 500 mL of RO water to 100 g of kiwi. "Orange 100% (made by Tokyo Meiraku Co., Ltd.)" for orange juice, "Apple 100% (made by Tokyo Meiraku Co., Ltd.)" for apple juice, and "One day's worth of vegetables (made by ITO EN Co., Ltd.)" for mixed vegetable juice. used.
Broccoli juice was prepared by incubating at 37 ° C. for 1 day after autoclaving.
The pH was adjusted by adding a 10N sodium hydroxide solution to pH 7 for kiwi juice and pH 6 for apple juice, orange juice and grapefruit juice.

To 50 mL of milk (made by Meiji Co., Ltd., delicious milk), glycerol stocks of three types of lactic acid bacteria were added, and anaerobic culture was carried out at 30 ° C. for one day. The viable cell count in milk was calculated by spreading 100 μL of a diluted solution of milk on an MRS agar medium, anaerobically culturing at 30 ° C., and measuring the number of colonies that appeared.
For the glycerol stock, each strain was spread on 0.5% calcium carbonate MRS agar medium and anaerobically cultured at 30 ° C. to form colonies. The colonies were inoculated into 14 mL of MRS liquid medium and anaerobically cultured at 30 ° C. for 1 day, and then the precipitate centrifuged at 8000 rpm and 4 ° C. for 5 minutes was suspended in 2 mL of 0.9% NaCl, and the like. It was mixed well with an amount of 80% glycerol solution and stored at -80 ° C.

<< Quantification of sugar concentration in vegetable / fruit juice >>
The total sugar concentration in the juice was quantified by the phenol-sulfuric acid method. The glucose concentration was quantified using Accu-Chek (manufactured by Roche).
The phenol-sulfuric acid method was performed as follows. The culture broth was centrifuged at 8000 rpm for 10 minutes, and 100 μL of the supernatant was collected. Next, 100 μL of 5% phenol was added and stirred vigorously with a vortex mixer for 5 seconds, 500 μL of sulfuric acid was added, and the mixture was vigorously stirred with a vortex mixer until heat was generated. After standing at room temperature for 20 minutes, OD490 was measured. Accucheck Aviva Trip F was set in Accucheck Aviva to quantify the glucose concentration.

Example 1
<Measurement of innate immunity promoting activity of each lactic acid bacterium>
The various isolated lactic acid bacteria were autoclaved, and after centrifugation, the bacterial cell component fraction was collected. The innate immunity promoting activity of each lactic acid bacterium was evaluated by injecting a suspension of the bacterium into a silk moth muscle specimen and measuring the contraction of the muscle. The results are shown in Table 1.
As a result, the specific activity of Leuconostoc carnosum # 7-2 (lactic acid bacterium # 7-2) was 460 units / mg, the specific activity of Leuconostoc gelidum # 4-2 (lactic acid bacterium # 4-2) was 250 units / mg, and Leuconostoc mesenteroides 8/11. The specific activity of -3 (lactic acid bacteria 8 / 11-3) was 250 units / mg, and high activity values were obtained for all lactic acid bacteria (Table 1).

Example 2
<Proliferation of each lactic acid bacterium in fruit / vegetable juice and milk>
Each of the above three lactic acid bacteria strains was inoculated into fruit / vegetable juice and milk, and the presence or absence of bacterial growth was examined. The results are shown in Tables 2-4.

Only lactic acid bacteria 8 / 11-3 were found to grow in unneutralized fruit and vegetable juices (Table 2). On the other hand, lactic acid bacteria # 7-2 and lactic acid bacteria # 4-2 proliferated when the fruit juice was neutralized (Table 3).
In addition, as a result of culturing each lactic acid bacterium in milk at 30 ° C. for 24 hours, proliferation was observed in each (Table 4).

Example 3
<Reduction of sugar content due to proliferation of each lactic acid bacterium in fruit / vegetable juice>
Lactic acid bacteria of each of the three strains were cultured in fruit juice, and the sugar concentration in the juice was quantified. The total sugar concentration was measured by the phenol sulfuric acid method. Glucose concentration was quantified by Accu-Chek (manufactured by Roche). The measurement results are shown in Table 5.

As a result of Table 5, the orange grapefruit juice in which lactic acid bacterium # 7-2 was grown, the orange grapefruit juice in which lactic acid bacterium # 4-2 was grown, and the kiwi grapefruit juice in which lactic acid bacterium 8 / 11-3 was grown were all included. A large decrease in sugar concentration and glucose concentration was observed.

The novel lactic acid bacterium of the present invention and the treated product have a high innate immunity activating ability, and further have an infectious disease preventive and therapeutic effect. Therefore, it is possible to provide drugs, foods and drinks containing innate immunity activators and infectious disease preventive and therapeutic agents that activate innate immunity using the lactic acid bacteria of the present invention, and are widely used in the pharmaceutical industry, food industry, etc. It is possible.

This application is based on Japanese Patent Application No. 2016-159557, which is a Japanese patent application filed on August 16, 2016, and the entire contents of those applications are cited herein as disclosure of the specification of the present invention. It is something that is taken in.

NITE BP-02307
NITE BP-02306
NITE BP-02308

SEQ ID NO: 1 is a base sequence corresponding to almost the entire length of 16SrDNA of an unknown strain (lactic acid bacterium # 7-2) belonging to the genus Leuconostoc.
SEQ ID NO: 2 is a base sequence corresponding to almost the entire length of 16SrDNA of an unknown strain (lactic acid bacterium # 4-2) belonging to the genus Leuconostoc.
SEQ ID NO: 3 is a base sequence corresponding to almost the entire length of 16S rDNA of an unknown strain (lactic acid bacterium 8 / 11-3) belonging to the genus Leuconostoc.

Claims (7)

A lactic acid bacterium belonging to the genus Leuconostoc whose accession number is NITE BP-02307 at the Patented Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE). A lactic acid bacterium belonging to the genus Leuconostoc whose accession number is NITE BP-02306 at the Patented Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE). A lactic acid bacterium belonging to the genus Leuconostoc whose accession number is NITE BP-02308 at the Patented Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE). An innate immune activator, wherein the lactic acid bacterium according to any one of claims 1 to 3 is autoclaved, and the bacterial cell component fraction recovered after centrifugation is used as an active ingredient . A food or drink containing the lactic acid bacterium according to any one of claims 1 to 3. A food or drink containing the innate immune activator according to claim 4. A food or drink produced by the step of fermenting with the lactic acid bacterium according to any one of claims 1 to 3.